CN110627927A - Shugeng sodium gluconate refining method for reducing purification loss rate - Google Patents
Shugeng sodium gluconate refining method for reducing purification loss rate Download PDFInfo
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Abstract
The invention discloses a sugammadex sodium refining method for reducing the product loss rate in the purification process, which purifies acidified sugammadex sodium by using magnetic polymer microspheres with PVA/mercapto group co-modified surfaces. The method has the advantages of high purification yield and purity, mild purification conditions, avoidance of side reactions, reduction of the usage amount of organic solvents, environment-friendly process, low purification cost and remarkably reduced purification loss rate.
Description
Technical Field
The invention belongs to the technical field of medicine purification, and particularly relates to a refining and purifying method of sugammadex sodium.
Background
The sugammadex sodium (sugammadex sodium, trade name Bridion) belongs to improved gamma cyclodextrin, is a chemical modifier of cyclodextrin, is also an antagonist of steroid non-depolarizing muscle relaxants, can form an inactive inclusion compound with steroid muscle relaxants, and antagonizes neuromuscular blockade at different depths. The sugammadex sodium is obtained by replacing all 6-hydroxyl groups in 8 glucopyranose units of gamma-cyclodextrin by carboxyethylthio. The synthesis method can be used for carrying out nucleophilic substitution reaction on 6-fully-deoxy-6-fully-halogenated-gamma-cyclodextrin and a 3-mercaptopropionic acid derivative under an alkaline condition to obtain the compound. WO0140316PP uses iodine as a halogenating reagent to react with gamma-cyclodextrin under the catalysis of triphenylphosphine to generate 6-deoxy-6-full iodo-gamma-cyclodextrin, the intermediate and 3-mercaptopropionic acid form thioether, and the target product is obtained after membrane dialysis and purification, but the purity is not high. WO2014125501(akzo nobel company) discloses a method for preparing sugammadex sodium, which comprises preparing halo-gamma-cyclodextrin by using a halophosphine reagent, and reacting the halo-gamma-cyclodextrin with 3-mercaptopropionic acid under the action of alkali metal alkoxide to form thioether so as to obtain a crude sugammadex sodium product; the crude product is dissolved in a mixture of water and methanol, and then is adsorbed by activated carbon, and then methanol is added for recrystallization to obtain sugammadex sodium crystals.
Structurally, the sugammadex sodium has a hollow cylinder structure, so that impurities such as inorganic salts, small molecular organic matters and the like are easily wrapped, and difficulty is brought to a refining method of a crude product of the sugammadex sodium.
CN105348412 discloses a purification method of a sugammadex sodium crude product, which comprises hydrolyzing the sugammadex sodium crude product under an acidic condition to obtain a free acid solid, pulping the free acid solid, washing and purifying the free acid solid; and reacting the free acid with organic amine to prepare the sugammadex ammonium salt, dissociating under an acidic condition to obtain the free acid, and reacting the obtained free acid with sodium hydroxide to prepare the pure sugammadex sodium product. However, only sugammadex sodium with a purity of 98% was obtained. CN 109021147A discloses a purification method of sugammadex sodium, which comprises the steps of dissociating crude sugammadex sodium into sugammadex acid in an organic acid-organic base-reducing agent mixed system, and further recrystallizing, purifying and salifying to obtain the sugammadex sodium. But this method introduces new impurities.
However, the above two methods have complicated steps, require multiple conversions between free acid and salt, are inconvenient to operate, and have large crystallization loss. In addition, since sugammadex is unstable in structure under acidic conditions, new impurities are easily generated by structural dissociation under acidic conditions, the yield is low, and the purity of the purified product is reduced.
WO2014125501 a1 reports a refining method of crude sugammadeca sodium, which removes impurities by a method of activated carbon adsorption and diatomite filtration, and then cools for recrystallization. CN107892727A discloses a purification method of a sugammadex sodium crude product, which adopts specific activated carbon for purification, and comprises the following steps: the method comprises the steps of pretreatment of activated carbon, dissolution of crude sugammadex sodium, purification of crude sugammadex sodium, dissolving the crude sugammadex sodium in water or a composition of water and a poor solvent of the sugammadex sodium, and adding the pretreated specific type of activated carbon into the solution to finally obtain the sugammadex sodium.
However, in the adsorption process of the activated carbon, part of the sugammadecase sodium and impurities are adsorbed in microporous pore canals of the activated carbon, so that the loss rate is high, and the product yield is not high. And the injection does not use active carbon, and the purification process of the active carbon is difficult to meet the requirement of medicinal production.
CN109553702A discloses a method for purifying sugammadex sodium, which uses a resin with single bonded tertiary amine anion functional group for purification, however, the method has not high purity and yield due to the difficult dissociation of resin adsorption and insufficient elution, and only sugammadex sodium with purity of 96% can be obtained with yield of 58%.
Therefore, due to the characteristics of easy degradation of a sugar unit of a cyclodextrin framework of sugammadex sodium when meeting strong acid, a molecular barrel structure and the like, the effective refining method of the crude sugammadex sodium is not more.
Aiming at the defects of the prior art, the method for refining the sugammadex sodium is needed to be provided, and the method has high yield and purity and mild purification conditions.
Disclosure of Invention
In view of the problems of the prior art, the invention aims to provide a method for purifying sugammadex sodium, which reduces the loss rate. The method has the advantages of simple operation, low cost and high product yield, can obviously improve the product purity, effectively inhibit the oxidation side reaction of sugammadex sodium in the purification process, has high material recycling degree, and is suitable for industrial production.
In order to achieve the purpose, the technical scheme mainly comprises the following steps.
(1) Dialyzing the crude sugammadex sodium to remove impurities, and removing small molecular impurities to obtain a sugammadex sodium prefabricated product;
(2) carrying out weak acidification treatment on the sugammadex sodium prefabricated product;
(3) adding magnetic polymer microspheres into the solution after the weak acidification treatment, and introducing nitrogen to stir under the atmosphere to perform adsorption reaction for 1-2 h;
(4) separating magnetic polymer microspheres by magnetic field, concentrating and crystallizing mother liquor, continuing to adsorb, dispersing polymer microspheres in deionized water, adjusting pH with sodium hydroxide solution, and performing shock desorption;
(5) and mixing the obtained sugammadex sodium solution, concentrating, adding ethanol or methanol, stirring and crystallizing to obtain refined sugammadex sodium.
The magnetic polymer microspheres are magnetic polymer microspheres with surfaces modified by PVA/sulfydryl groups.
Specifically, the detailed technical scheme of the invention is as follows.
A method for refining sugammadex sodium based on polymer microspheres comprises the following steps:
step (1): dialysis impurity removal pretreatment:
dissolving the crude product of sugammadex sodium with deionized water, dialyzing with dialysis bag in distilled water at 40-50 deg.C for 1-3 times (60-90 min each time) to remove small molecular impurities such as unreacted raw materials; and (3) after dialysis, placing in an ice bath for cooling, cooling and crystallizing by using ethanol or acetonitrile solvent with 5-10 times of volume, filtering after crystallization is finished, and collecting white solid, namely the sugammadex pre-product.
The purity of the crude sugammadex sodium crystallized in the step can reach 95-98%.
In this step, the cut-off molecular weight of the dialysis bag is 1000-1500 Da.
In this step, the ethanol or acetonitrile solvent may be replaced by other poor solvents such as acetone, ethyl acetate, methanol, etc.
Step (2): weak acidification treatment:
placing the dialyzed sugammadex sodium prefabricated product into a flask with a stirrer, stirring and dissolving the sugammadex sodium prefabricated product by using 15-30 times of deionized water at 35-50 ℃, and dropwise adding citric acid or acetic acid to adjust the pH value of the solution to 6.0-6.8.
Preferably, the pH of the solution is adjusted to 6.2-6.5.
Preferably, deionized water is added to the preform in an amount of 15 to 25 times the mass of the preform.
In this step, citric acid or acetic acid may be replaced by other organic acids, such as formic acid.
And (3): adsorption treatment:
adding PVA/sulfydryl group co-modified magnetic polymer microspheres to the acidified solution to 1-10 wt%, and introducing nitrogen to replace the atmosphere; maintaining the temperature of the water bath at 50-55 ℃, slowly stirring and reacting for 1-2h, so that the sugamonic acid and the polymer microspheres are fully adsorbed and grafted on the surfaces of the magnetic polymer microspheres.
Preferably, the addition amount of the magnetic polymer microspheres is 3-5 wt% of the solution.
Under a weakly acidic environment, a large number of free polar hydroxyl groups on PVA on the polymer microsphere can perform adsorption reaction with carboxyl and other groups on the surface of the sugamonic acid molecule through the actions of hydrogen bonds, esterification and the like, so that the PVA is grafted on the surface of the magnetic polymer microsphere; in addition, the surface sulfhydryl group on the polymer microsphere can further enhance the adsorption effect by coordinating with the surface atom of the sugammadecaonic acid molecule, and the sulfhydryl group also maintains the reducing environment on the microsphere adsorption interface, thereby avoiding the defect that the sugammadecane sodium molecule is easy to be oxidized to generate by-products due to the existence of thioether bonds.
Meanwhile, the mesoporous silica coated on the surface of the polymer microsphere can not only assist the adsorption effect through surface atom coordination, but also further adsorb small molecular impurities which are not removed through dialysis in the inner pores.
And (4): solid-liquid separation treatment:
cooling the reaction system after the microsphere adsorption is finished, and putting the obtained mixed solution into an electromagnetic separation device to be electrified with a magnetic field so as to separate the magnetic polymer microspheres; concentrating the mother liquor after microsphere separation, adding acetone for crystallization, collecting unadsorbed residual sugamonic acid solid, taking the unadsorbed residual sugamonic acid solid as a circulating raw material, combining the circulating raw material with the next batch of crude sugamonic acid sodium raw material to be adsorbed, and reserving for further adsorption treatment.
Optionally, the mother liquor after microsphere separation is added into new polymer microspheres for secondary or multiple adsorption, and the separated polymer microspheres are combined.
And (5): desorption:
s1: dispersing the polymer microspheres separated by the magnetic field in a proper amount of deionized water at 25-40 ℃, adjusting the pH value to 8-9 by using a sodium hydroxide solution, carrying out vibration desorption for 30-60min, and then separating the polymer microspheres by passing the magnetic field;
s2: continuously oscillating and desorbing the separated polymer microspheres in alkali liquor, and repeating desorption operation until the separated mother liquor does not contain sugammadex sodium molecules; the desorbed polymer microspheres are ultrasonically washed by deionized water and absolute ethyl alcohol and then are repeatedly utilized;
s3: the desorbed solutions were combined to give a pale yellow transparent sugammadex sodium solution.
In this step, the criterion of substantially no sugammadex molecules may be set as appropriate, for example, less than 1%, less than 0.5%, less than 0.3%, etc. of sugammadex in the mother liquor.
And (6): and (3) post-treatment:
and (3) carrying out vacuum evaporation concentration on the obtained sugammadex sodium solution, placing the solution into an ice bath for cooling after the solution is concentrated to a proper volume (for example, 10-50% of the original volume), adding 5-10 times of ethanol or methanol into the solution at the same time, stirring and crystallizing for 0.5-2h, carrying out suction filtration after crystallization is finished, washing the obtained white solid with methanol, and carrying out vacuum drying to obtain the refined sugammadex sodium.
In this step, crystallization is not performed by vacuum evaporation to dryness, so as to avoid the concomitant precipitation of impurities such as residual sodium hydroxide and sodium chloride.
In the invention, the PVA/sulfhydryl group co-modified magnetic polymer microsphere is prepared by the following steps.
S1：Fe3O4Preparation of core particles:
at 50 ℃, FeCl of 0.1-0.5mol/L is added2Solution and FeCl3Adding the solution into a three-neck flask according to the molar ratio of 1:2, and uniformly stirring and mixing; introducing nitrogen for replacement, dropwise adding 25-30 wt% of ammonia water into the flask by using a dropping funnel under the nitrogen atmosphere, and stirring while dropwise adding until the pH value of the system is 10-12; heating in water bath to 55-60 deg.C, continuously stirring for 45-60min, centrifuging to obtain precipitate, repeatedly washing with distilled water to neutrality, oven drying, and grinding to obtain powdered Fe3O4A core particle.
S2: preparing mesoporous silica coated magnetic microspheres:
mixing Fe3O4Dispersing the nanometer particles in deionized water, adjusting the pH value to 9.0-9.5 with sodium hydroxide solution, and heating to 55-60 deg.C in water bath; adding tetraethyl orthosilicate and ethyl acetate under the stirring state, uniformly stirring, adding an MPS coupling agent (3- (trimethoxysilyl) propyl-2-methyl-2-acrylate), keeping the reaction temperature, stirring and reacting for 16-24 hours, centrifuging after the reaction is finished, taking the precipitate, and fully washing with ethanol to obtain the magnetic microsphere with the surface coated with the mesoporous silica, wherein the particle size is 200-500 nm.
S3: co-modifying the surface of thiol group and PVA:
heating and swelling PVA particles into 5-10 wt% solution by using distilled water, pouring magnetic microspheres coated with mesoporous silica into the PVA solution, adding DMF-n-butanol dispersion solution with the mass of 6-10 times that of the magnetic microspheres for dispersion, adding a proper amount of mercaptopropyl trimethoxy silane after uniform dispersion, stirring and reacting for 4-6h at the reflux temperature, centrifugally separating and precipitating, washing by using absolute ethyl alcohol and distilled water in sequence, and drying to obtain the PVA/mercapto group co-modified magnetic polymer microspheres.
Wherein the ratio of DMF in the dispersion solution: the volume ratio of the n-butanol is 1: 1-2.
Wherein the using amount of the mercaptopropyl trimethoxy silane is 1-1.5 times of the mass of the magnetic microspheres.
Wherein the dosage of the PVA is 1-2 times of the mass of the magnetic microspheres.
On the other hand, the invention also provides a refined sugammadex sodium product prepared by the method.
The technical effects of the invention include but are not limited to the following aspects:
(1) in the invention, a weak acidic environment is constructed to acidify sugammadex sodium, and a large number of free polar hydroxyl groups on silicon dioxide and PVA on the polymer microspheres can perform physical and chemical adsorption reaction with carboxyl groups and other groups on the surface of sugammadex molecules through the actions of hydrogen bonds, esterification and the like. Reversible adsorption is carried out through the surface adsorption effect of the magnetic polymer microspheres and the sodium sugammadex molecules, and mother liquor residue is repeatedly recovered, so that on the basis of improving the product purity, compared with purification methods such as crystallization method purification and activated carbon, the loss rate can be effectively and obviously reduced, and the yield of a refined product is improved (the yield based on a prefabricated product is higher than 85%, and the yield based on a crude raw material is higher than 70%).
(2) The polymer microsphere is modified by a silane coupling agent containing reducing sulfhydryl groups, so that the surface sulfhydryl groups on the polymer microsphere not only can further enhance the adsorption effect by coordinating with surface atoms of sugammadex acid molecules, but also the sulfhydryl groups can maintain the reducing environment on the adsorption interface of the microsphere, and the defect that the sugammadex sodium molecules are easily oxidized to generate byproducts due to the existence of thioether bonds is avoided. Compared with the prior art, the addition of the reducing agent or the oxidation inhibitor avoids the introduction of a new pollution source, facilitates the post-treatment, does not discharge waste water and liquid, and is green and environment-friendly.
(3) The mesoporous silica coated on the surface of the polymer microsphere can not only assist the adsorption effect through surface atom coordination, but also can further adsorb small molecular impurities which are not removed by dialysis through the pores in the mesoporous silica, and the sugammadex sodium is difficult to enter the mesoporous channel due to the existence of macromolecules such as PVA on the surface; the desorbed polymer microspheres can be washed by ultrasonic vibration to effectively remove the small molecular impurities adsorbed inside, so that the polymer microspheres are convenient to recycle.
(4) Compared with an inorganic strong acid environment, the method disclosed by the invention has the advantages that the weak acid environment is adopted for acidification treatment, the defects that sugar units of a cyclodextrin framework of the sugammadex sodium are degraded when meeting strong acid and glycosidic bonds are easy to open to generate new byproducts are avoided, the sulfydryl reducing group environment of a microsphere adsorption interface is matched, the oxidation of the sugammadex sodium is reduced to the maximum extent, and the generation of new impurities is inhibited.
(5) The invention mainly adopts deionized water as a reaction solvent, greatly reduces the use amount of an organic solvent, has simple and convenient process operation, has conventional and cheap reagents, can regenerate and reuse the polymer microspheres, and is beneficial to industrialized low-cost production.
Drawings
FIG. 1 TEM photograph (2000) of magnetic polymer microsphere prepared in preparation example 2
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Preparation example 1
Preparation of crude sugammadex sodium
Under the protection of nitrogen, 11.8g of sodium hydride is added in portions to 550ml of DMF; then, a solution of 3-mercaptopropionic acid (15.8g) in 100ml of DMF solvent was added dropwise under ice bath. After the dropwise addition, stirring for 15 minutes at room temperature, then heating to 70 ℃, and slowly dropwise adding 150ml of DMF (dimethyl formamide) solution of iodo gamma-cyclodextrin under the stirring condition, wherein 20.1g of iodo gamma-cyclodextrin is contained; the reaction was stirred for 8h with the temperature maintained. After the reaction is finished, the reaction system is cooled to room temperature in an ice bath, 150ml of deionized water is added, and the reaction is continuously stirred for 2 hours. After the reaction, the reaction solution is decompressed and concentrated to 260ml, then the solution is cooled in an ice bath, 1L of methanol is added, the solution is stirred and crystallized, the solution is filtered, the solid is collected, and the solid is dried to obtain 25.3g of crude sugammadex sodium with the HPLC purity of 91.03%.
Preparation example 2
Preparation of PVA/sulfydryl group co-modified magnetic polymer microspheres
(1) At 50 ℃, 0.4mol/L FeCl is added2Solution and FeCl3Adding the solution into a three-neck flask according to the molar ratio of 1:2, and uniformly stirring and mixing to obtain 240ml of mixed solution; introducing nitrogen for replacement, dropwise adding 28 wt% ammonia water into the flask by using a dropping funnel under the nitrogen atmosphere, and stirring while dropwise adding until the pH value of the system is 11; heating in water bath to reaction temperature of 57 deg.C, continuously stirring for reaction for 60min, centrifuging to obtain precipitate after reaction, repeatedly washing with distilled water to neutrality, oven drying, and grinding to obtain powdered Fe3O4A core particle.
Taking the above Fe3O4Dispersing 8g of nuclear particles in 800mL of deionized water, adjusting the pH value to 9.5 by using 1M sodium hydroxide solution, and heating to 55 ℃ in a water bath; adding 180mL of tetraethyl orthosilicate and 480mL of ethyl acetate under the stirring state, uniformly stirring, adding 20g of MPS coupling agent, keeping the reaction temperature, stirring for reacting for 16 hours, centrifuging after the reaction is finished, taking the precipitate, and fully washing with ethanol to obtain the magnetic microspheres with the surfaces coated with the mesoporous silica, wherein the particle size distribution is 200-500 nm.
(3) Heating and swelling 8g of PVA particles by using 92g of distilled water, pouring 5g of the mesoporous silica-coated magnetic microspheres into the PVA solution, adding 60ml of DMF-n-butanol dispersion solution (wherein DMF: n-butanol is 1:2) for dispersion, adding 10.5g of mercaptopropyl trimethoxy silane after uniform dispersion, stirring and reacting for 6h at the reflux temperature, then centrifugally separating and precipitating, washing the obtained precipitate with absolute ethyl alcohol and distilled water in sequence, and drying in vacuum to obtain the PVA/mercapto group co-modified magnetic polymer microspheres with the particle size of less than 1 mu m.
The prepared polymer microspheres are subjected to electron microscope observation and characterization, and a TEM picture is shown in FIG. 1.
Example 1
1) Taking 11.8g of the prepared sugammadex sodium raw material, heating and dissolving the raw material with 60mL of deionized water, placing the raw material into a dialysis bag with the molecular weight cutoff of 1200Da, dialyzing the raw material in distilled water at 45 ℃ for 2 times, and removing small molecular impurities for 60min each time; and (3) after dialysis, placing the mixture in a beaker for ice bath cooling, simultaneously stirring the mixture by using 680mL of acetonitrile solvent for crystallization, filtering the mixture after the crystallization is finished, collecting white solid, drying the white solid by 10.2g, namely the prefabricated crude product of the sugammadex sodium, wherein the HPLC purity is 95.62%.
2) 10.0g of the dialyzed premade crude sugammadex sodium product was placed in a three-neck flask with a stirrer, dissolved with 290mL of deionized water at 50 ℃ under stirring, and the pH of the solution was adjusted to 6.2 by dropwise addition of acetic acid.
3) Adding 15g of PVA/sulfydryl group co-modified magnetic polymer microspheres prepared according to the method into the acidified solution, and introducing nitrogen to replace air atmosphere; maintaining the temperature of the water bath at 55 ℃, slowly stirring and reacting for 2h, so that the sugamonic acid and the polymer microspheres are fully adsorbed and grafted on the surfaces of the magnetic polymer microspheres.
4) Cooling the reaction system to room temperature after adsorption is finished, placing the obtained mixed solution in a separator of a charged magnetic field generating device, and carrying out solid-liquid separation by passing a magnetic field so as to separate out the magnetic polymer microspheres; adding new magnetic polymer microspheres into the mother liquor after microsphere separation to 3 wt%, continuously stirring and adsorbing for 1h, and then carrying out solid-liquid separation and combining the separated polymer microspheres.
And concentrating the residual mother liquor to 60ml, adding acetone on an ice bath for cooling and crystallizing, collecting unadsorbed residual sugamluconic acid solid, combining the unadsorbed residual sugamluconic acid solid serving as a circulating raw material with the next batch of crude sugamluconate raw material to be adsorbed, and reserving for further adsorption treatment.
5) Dispersing the separated polymer microspheres in 450mL of 40 ℃ deionized water, adjusting the pH to 9 by using a 2M sodium hydroxide solution, carrying out vibration desorption on a shaking table for 30min, and then carrying out magnetic field solid-liquid separation to separate the polymer microspheres; dispersing the separated polymer microspheres in 250mL of deionized water again, adjusting the pH value to 9 by using sodium hydroxide, and continuing to vibrate for desorption; repeatedly desorbing until mother liquor obtained after microsphere separation does not contain sugammadex molecules basically; and (3) ultrasonically washing the polymer microspheres with deionized water and absolute ethyl alcohol, and then, reserving for recycling.
The desorbed solutions were combined to give about 900mL of a pale yellow, clear solution of sugammadex sodium.
6) And (3) carrying out vacuum evaporation and concentration on the obtained sugammadex sodium solution to about 120mL, taking out, placing in an ice bath for cooling, adding 850mL of absolute ethyl alcohol at the same time, stirring and crystallizing for 0.5h, carrying out suction filtration after crystallization, washing the obtained white solid with methanol, and carrying out vacuum drying to obtain 9.1g of refined sugammadex sodium with the HPLC purity of 99.68%.
The purification yield was about 77% based on the crude sugammadex sodium starting material; the purification yield was about 91% based on the preformed crude sugammadex sodium.
Example 2
1) Taking 12g of sugammadex sodium prepared in the preparation example, heating and dissolving the raw material by 60mL of deionized water, placing the raw material into a dialysis bag with the molecular weight cutoff of 1500Da, dialyzing the solution in distilled water at 45 ℃ for 2 times, and removing small molecular impurities for 60min each time; and (3) after dialysis, placing the mixture in a beaker for ice bath cooling, simultaneously stirring the mixture by using 700mL of acetonitrile solvent for crystallization, filtering the mixture after the crystallization is finished, collecting white solid, drying and drying the white solid by 10.5g, namely the prefabricated crude product of the sugammadex sodium, wherein the HPLC purity is 95.36%.
2) 10.0g of the prepared crude sugammadex sodium is placed in a three-neck flask with a stirrer, 290mL of deionized water is used for stirring and dissolving at 50 ℃, and acetic acid is added dropwise to adjust the pH value of the solution to 6.6.
3) Adding 15g of PVA/sulfydryl group co-modified magnetic polymer microspheres prepared according to the method into the acidified solution, and introducing nitrogen to replace air atmosphere; maintaining the temperature of the water bath at 55 ℃, slowly stirring and reacting for 2h, so that the sugamonic acid and the polymer microspheres are fully adsorbed and grafted on the surfaces of the magnetic polymer microspheres.
4) Cooling the reaction system to room temperature after adsorption is finished, and introducing the obtained mixed solution into a magnetic field for solid-liquid separation so as to separate out the magnetic polymer microspheres; and concentrating the residual mother liquor to about 60ml, placing the concentrated mother liquor on an ice bath, adding acetone for cooling and crystallizing, collecting residual sugamluconic acid solid, combining the residual sugamluconic acid solid serving as a circulating raw material with the next batch of crude sugamluconic acid sodium raw material to be adsorbed, and keeping the mixture for continuous adsorption treatment.
5) Dispersing the separated polymer microspheres in 400mL of deionized water at 40 ℃, adjusting the pH to 8.8 by using 1M sodium hydroxide solution, oscillating and desorbing on a shaking table for 30min, and starting an electromagnetic device to separate solid from liquid by using a magnetic field so as to separate the polymer microspheres; dispersing the separated polymer microspheres in 200mL of deionized water, adjusting the pH value to 8.8 by using sodium hydroxide, continuing to vibrate for desorption, and repeatedly performing desorption until mother liquor after the microspheres are separated does not contain sugammadex sodium molecules basically; and (3) ultrasonically washing the polymer microspheres with deionized water and absolute ethyl alcohol, and then, reserving for recycling.
The desorbed solutions were combined to give about 780mL of a pale yellow, clear solution of sugammadex sodium.
6) And (3) carrying out vacuum evaporation and concentration on the obtained sugammadex sodium solution to about 100mL, taking out, placing in an ice bath for cooling, adding 800mL of methanol at the same time, stirring and crystallizing for 1h, carrying out suction filtration after crystallization is finished, washing the obtained white solid with methanol, and carrying out vacuum drying to obtain 8.7g of refined sugammadex sodium, wherein the HPLC purity is 99.73%, and the total content of impurities is lower than 0.3%.
The purification yield was about 73% based on the crude sugammadex sodium raw material; the purification yield was about 87% based on the preformed crude sugammadex sodium.
Comparative example 1
1) Crude sugammadex preparation was prepared from sugammadex starting material as in example 2.
2) 10.0g of the pre-prepared crude sugammadex sodium was placed in a three-necked flask with stirring and dissolved in 290mL of deionized water at 50 ℃ with stirring.
3) Adding 15g of activated carbon into the solution, and introducing nitrogen to replace air atmosphere; the temperature of the water bath is maintained at 55 ℃, and the reaction is slowly stirred for 2 hours.
4) The reaction system is filtered while hot, and the filtrate is cooled to room temperature.
5) And (3) carrying out vacuum evaporation and concentration on the obtained sugammadex sodium solution to about 100mL, taking out, placing in an ice bath for cooling, adding 800mL of methanol at the same time, stirring and crystallizing for 1h, carrying out suction filtration after crystallization is finished, and carrying out vacuum drying to obtain 6.6g of refined sugammadex sodium, wherein the HPLC purity is 99.15%, and the total content of impurities is close to 0.9%.
The purification yield was about 55% based on the crude sugammadex sodium raw material; the purification yield was about 66% based on the preformed crude sugammadex sodium.
Comparative example 2
1) Crude sugammadex preparation was prepared from sugammadex starting material as in example 2.
2) 10.0g of the preformed crude sugammadex sodium product was placed in a three-necked flask with stirring, dissolved with 290mL of deionized water at 50 ℃ with stirring, and the solution pH was adjusted to 6.6 by dropwise addition of acetic acid.
3) Adding 5g of activated carbon into the solution, and introducing nitrogen to replace air atmosphere; the temperature of the water bath is maintained at 55 ℃, and the reaction is slowly stirred for 1 h.
4) The reaction system was filtered while hot, the filtrate was cooled to room temperature, and the pH was adjusted to 8.8 with 1M sodium hydroxide solution.
5) And (3) carrying out vacuum evaporation and concentration on the obtained sugammadex sodium solution to about 100mL, taking out, placing in an ice bath for cooling, adding 800mL of methanol at the same time, stirring and crystallizing for 1h, carrying out suction filtration after crystallization, and carrying out vacuum drying to obtain 7.1g of refined sugammadex sodium with the HPLC purity of 99.02%.
The purification yield is about 60% based on the crude sugammadex sodium raw material; the purification yield was about 71% based on the preformed crude sugammadex sodium.
According to the comparative example, the purification method can effectively reduce the loss in the purification process on the basis of ensuring that the purification purity reaches 99 percent, and the total product yield is higher; considering that a part of the product lost is distributed in the mother liquor and recycled (the part of the recovered product is not calculated in the actual yield), the actual loss rate is lower.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A refining method of sugammadex sodium is characterized by comprising the following steps:
(1) carrying out dialysis pretreatment on the sugammadex sodium crude product to remove small molecular impurities to obtain a sugammadex sodium prefabricated product;
(2) carrying out weak acidification treatment on the sugammadex sodium prefabricated product;
(3) adding magnetic polymer microspheres into the solution after the weak acidification treatment, and stirring the solution in a nitrogen atmosphere to perform an adsorption reaction for 1 to 2 hours;
(4) separating out magnetic polymer microspheres, dispersing the polymer microspheres in deionized water, adjusting the pH value by using a sodium hydroxide solution, oscillating for desorption, and carrying out solid-liquid separation to obtain a sugammadex sodium solution;
(5) mixing the obtained sugammadex sodium solution, concentrating, adding ethanol or methanol, stirring and crystallizing to obtain refined sugammadex sodium;
preferably, the magnetic polymer microspheres are magnetic polymer microspheres with surfaces co-modified by PVA/sulfhydryl groups.
2. The refining method of sugammadex sodium is characterized by comprising the following specific steps:
(1) dissolving the crude sugammadex sodium with deionized water, and dialyzing in distilled water at 40-50 deg.C for 1-3 times to remove small molecular impurities; placing the mixture in an ice bath for cooling after dialysis, cooling and crystallizing by using an ethanol or acetonitrile solvent, and filtering and collecting white solid to obtain a sugammadex prefabricated product;
(2) placing the dialyzed sugammadex sodium prefabricated product into a flask with a stirrer, stirring and dissolving the sugammadex sodium prefabricated product by using deionized water with the mass of 15-30 times of the mass of the sugammadex sodium prefabricated product at 35-50 ℃, and dropwise adding citric acid or acetic acid to adjust the pH value of the solution to 6.0-6.8;
(3) adding PVA/sulfydryl group co-modified magnetic polymer microspheres to the acidified solution to 1-10 wt%, and introducing nitrogen to replace the atmosphere; maintaining the temperature of the water bath at 50-55 ℃, and slowly stirring for reaction for 1-2h to ensure that the sugamonic acid and the polymer microspheres are fully adsorbed;
(4) cooling the reaction system after adsorption is finished, and putting the obtained mixed solution into an electromagnetic separation device to be electrified with a magnetic field so as to separate the magnetic polymer microspheres; concentrating the mother liquor after microsphere separation, adding acetone for crystallization, collecting unadsorbed residual sugamonic acid solid, taking the unadsorbed residual sugamonic acid solid as a circulating raw material, combining the circulating raw material with the next batch of crude sugamonic acid sodium raw material to be adsorbed, and reserving the mixture for adsorption treatment;
(5) and (3) desorption treatment:
s1: dispersing the polymer microspheres separated by the magnetic field in a proper amount of deionized water at 25-40 ℃, adjusting the pH of the solution to 8-9 by using sodium hydroxide, and separating the polymer microspheres after shaking desorption for 30-60 min;
s2: continuously oscillating and desorbing the separated polymer microspheres in alkali liquor, and repeating desorption operation until the separated mother liquor does not contain sugammadex sodium molecules; the desorbed polymer microspheres are ultrasonically washed by deionized water and absolute ethyl alcohol and then are repeatedly utilized;
s3: combining the desorption solutions to obtain a light yellow transparent sugammadex sodium solution;
(6) and (3) carrying out vacuum evaporation concentration on the obtained sugammadex sodium solution, placing the solution into an ice bath for cooling after the solution is concentrated to a proper volume, adding ethanol or methanol into the solution at the same time, stirring the solution for crystallization for 0.5 to 2 hours, carrying out suction filtration after the crystallization is finished, washing the obtained white solid with methanol, and carrying out vacuum drying to obtain the refined sugammadex sodium.
3. The method according to claim 2, wherein in the step (2), the pH of the solution is adjusted to 6.2 to 6.5.
4. The method of claim 2, wherein in the step (3), the magnetic polymer microspheres are added in an amount of 3-5 wt% of the solution.
5. The method of claim 2, wherein the step (4) further comprises adding the mother liquor after the separation of the microspheres to new polymeric microspheres for secondary adsorption, and combining the separated polymeric microspheres.
6. The method according to claim 2, wherein in the step (6), 5 to 10 times by volume of ethanol or methanol is added to perform the crystallization.
7. The method according to claim 1 or 2, wherein the PVA/thiol group co-modified magnetic polymer microsphere is prepared by the following steps:
s1: at 50 ℃, FeCl of 0.1-0.5mol/L is added2Solution and FeCl3Adding the solution into a three-neck flask according to the molar ratio of 1:2, and uniformly stirring and mixing; introducing nitrogen for replacement, dropwise adding 25-30 wt% of ammonia water into the flask by using a dropping funnel under the nitrogen atmosphere, and stirring while dropwise adding until the pH value of the system is 10-12; heating in water bath to 55-60 deg.C, continuously stirring for 45-60min, centrifuging to obtain precipitate, repeatedly washing with distilled water to neutrality, oven drying, and grinding to obtain powdered Fe3O4A core particle;
s2: mixing Fe3O4Dispersing the nanometer particles in deionized water, adjusting the pH value to 9.0-9.5 with sodium hydroxide solution, and heating to 55-60 deg.C in water bath; adding tetraethyl orthosilicate and ethyl acetate under the stirring state, stirring uniformly, adding MPS coupling agent, keeping the reaction temperature, stirring and reacting for 16-24 hours, centrifuging after the reaction is finished, taking precipitate, and fully washing with ethanol to obtain Fe with the surface coated with mesoporous silica3O4Magnetic microspheres;
s3: heating and swelling PVA particles into 5-10 wt% solution by using distilled water, pouring magnetic microspheres coated with mesoporous silica into the PVA solution, adding DMF-n-butanol dispersion solution with the mass of 6-10 times that of the magnetic microspheres for dispersion, adding a proper amount of mercaptopropyl trimethoxy silane after uniform dispersion, stirring and reacting for 4-6h at the reflux temperature, centrifugally separating and precipitating, washing by using absolute ethyl alcohol and distilled water in sequence, and drying to obtain the PVA/mercapto group co-modified magnetic polymer microspheres.
8. The method of claim 7, wherein the ratio of DMF in the dispersion solution: the volume ratio of the n-butanol is 1: 1-2; the using amount of the mercaptopropyl trimethoxy silane is 1-1.5 times of the mass of the microspheres; the amount of PVA is 1-2 times of the mass of the microspheres.
9. Sugammadex sodium prepared according to the process of any one of claims 1 to 6.
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